The ARF tumor suppressor protein induces a p53 response, resulting in cell cycle arrest or apoptotic cell death through signaling pathways that remain to be precisely elucidated. Certain small DNA viruses activate the ARF pathway but also inactivate the downstream p53 function to prevent apoptosis/cell cycle arrest. Polyoma virus (PY) is unusual with respect to other small DNA viruses, in that none of its early region proteins interact directly with the p53 protein. It would be expected that PY would also suppress the generation of active p53 and the main focus of the proposed research is to understand what mechanisms PY uses to interfere with this process. The complete PY early region encoding the large T-antigen (PYLT), middle T-antigen (PYMT) and small T-antigen (PYST) will transform primary rodent cells and REF52 cells. PYMT, the main PY oncogene, by itself, will transform these cells only when p53 or ARF is inactivated. We find that PYMT alone activates an ARE-induced p53 mediated block of REF52 cell division and propose that this explains the inability of PYMT alone to transform p53+/ARF+ cells. In PY transformed REF52 cells, where all three PY T-antigens are present, ARF is still upregulated but p53 is not activated. Thus, PYLT and/or PYST can negate the ARF-induced block to cell division induced by PYMT resulting in transformed cells. We intend to use the PY inhibition of ARF signaling to p53 to better define the important ARF-p53 tumor suppressor pathway, as well as to provide a better understanding of oncogene co-operation. Our hypothesis is that PY is revealing an important new aspect of the ARF-p53 tumor suppressor signaling circuit, and we plan to use PY as a tool to study its molecular basis. The specific aims of this proposal are: 1) To identify which PY protein(s) (and domains) are involved in inhibiting ARF signaling by assessing the ability of wild type and mutant PY proteins to overcome an ARF-induced p53 block to cell division. 2) To investigate the mechanism of inhibition of ARF signaling by assessing differences between both the ARF protein and the p53 regulator protein MDM2, and their complexes, from cells in which ARF is successfully signaling to p53 and cells in which PY inhibits the ARF signal to p53. 3) To determine if MDM2 can overcome the ARF-induced p53 block and complement PYMT for generation of dividing transformed cells and to use genetic screens to identify unrecognized cellular genes that can negate a PY-induced ARF-mediated p53 block to cell division. These aims will collectively serve to characterize a novel property of the ARF-p53 tumor suppressor signaling circuit, and potentially suggest new therapeutic approaches to restoring p53 function and exploiting p53 dysfunction in cancer cells.